Process for the decomposition of acrylamide

ABSTRACT

A process for the decomposition of acrylamide using an amidase (acrylamide amido hydrolase) enzyme in which the enzyme is induced in strains of Methylophilus methylotrophus. A method for inducing the enzyme and a process for producing acrylic acid are also claimed. The process for the decomposition of acrylamide is useful in particular for reducing the level of unreacted monomer associated with homo- and hetero- polymers of acrylamide. The activity of the enzyme can be increased by heating it to a temperature in the range 40° to 80° C.

This invention relates to a process for the decomposition of acrylamide,to a method for the production of the enzyme amidase (acylamide amidohydrolase E.C. No. 3-5-1-4) used in the decomposition of acrylamide andto a process for the production of acrylic acid or a salt or esterthereof by decomposition of acrylamide.

Polyacrylamide polymers, i.e. homo- and hetero- polymers of acrylamide,are widely used as flocculants in the potable water industry, sewagetreatment, paper manufacture and mining. However their utility isrestricted and they cannot for instance be used in connection withfoodstuffs because they are generally contaminated with unreactedacrylamide monomer which is a cumulative neurotoxin and a carcinogen. Atpresent polyacrylamides are generally allowed in USA to contain up to500 ppm of unreacted acrylamide and this limit is likely to beintroduced elsewhere. This level of unreacted monomer can be achieved byuse of longer polymerisation times and by a "heat treating" process.Such treatments increase cost of manufacture and reduce the efficiencyof the polymers produced by causing branching in these polymers. Linearpolymers have a higher flocculation efficiency and are preferred. Thereis a need for a reliable process for reducing the level of unreactedmonomer in polyacrylamides without damage to the polymer to 500 ppm andpreferably to even lower levels (5-10 ppm or lower). If the unreactedmonomer present could be reduced reliably to very low levels (1-5 ppm orless) polyacrylamides could be considered for uses from which they arepresently excluded, e.g. in the manufacture of food contact materials orin direct contact with food.

Japanese Patent Publication No. 79011389 (corresponding to JapaneseKokai No. 53086078) discloses a process for the decomposition ofacrylamide monomer, e.g. in waste water or in polyacrylamides bycontacting with an intracellular enzyme of Brevibacterium ammoniogenespreferably of strains ATCC 1641, ATCC 6871 and ATCC 6872. However,despite being known for a number of years, this process does not appearto have been used commercially to any significant extent. MoreoverBrevibacterium ammoniogenes enzyme works poorly in polyacrylamide latexsystems.

According to a first aspect of the present invention we provide aprocess for the decomposition of acrylamide in a medium containing it inwhich the medium is contacted with an enzyme capable of decomposingacrylamide under conditions suitable for the enzyme to decompose theacrylamide wherein the enzyme is an amidase enzyme which has beeninduced in a bacterium belonging to the species Methylophilusmethylotrophus.

Further according to the invention we provide a method for theproduction of an amidase enzyme wherein a bacterium belonging to thespecies Methylophilus methylotrophus is cultivated in a mediumcontaining appropriate nutrients and an amide under conditions such thatthe amidase enzyme is induced in the bacterium. The modified bacteriumproduced in the method of the invention is also included in the scope ofthe invention.

Further according to the invention we provide a process for theproduction of acrylic acid or a salt or ester thereof in which a mediumcontaining acrylamide is contacted with an enzyme capable of decomposingacrylamide to acrylic acid under conditions suitable for the enzyme todecompose the acrylamide to acrylic acid or a salt or ester thereofwherein the enzyme is an amidase enzyme which has been induced in abacterium belonging to the species Methylophilus methylotrophus.

Preferably the method of the invention is used to induce an amidaseenzyme capable of decomposing acrylamide in the bacterium which enzymemay be used in the processes of the invention. Hereinafter the inventionwill be described mainly in terms of the production and use of thisenzyme.

Amidases are part of the nitrogen assimilation apparatus of many cellsgrown under conditions where the sole source of nitrogen is an aliphaticamide.

Thus the metabolic role of amidase is to release ammonia from saidamide, e.g. by the following reaction: ##STR1##

The ammonia released is then assimilated into protein biosynthesis. Thealiphatic acid produced may or may not be assimilated dependent upon themicroorganism type.

Amidases can also catalyse the conversion of acrylamide to acrylic acidby the following reaction: ##STR2##

In the presence of appropriate salts or alcohols the acrylic acidproduced can be converted to its salt or ester.

The decomposition process of the invention can be used to decomposeacrylamide in any medium in which it occurs. It is particularly usefulfor decomposing unreacted acrylamide present in polyacrylamide polymersand acrylamide present in waste waters, e.g. waste water from a processfor the production of polyacrylamides. Polyacrylamides are produced aspolymers of three types i.e. solution, dry and suspension polymers.

Polyacrylamides are produced as polymers of three chemical types. Theseare cationic, anionic and nonionic polymers wherein acrylamides may becopolymerised with other monomers, e.g. acrylic acid. These polymers maybe manufactured by one of the following three basic technologies:

Solution polymerisation wherein monomers are polymerised in aqueoussolution to produce a gel-type product;

Dry polymers which are polymers produced as above but which aresubsequently heat dehydrated prior to use;

Latex or suspension polymerisation wherein a solution of monomers inwater is admixed with detergents and a non-aqueous low odor paraffinicsolvent to form a stable suspension of aqueous droplets within whichbeads of polymers are formed by addition of a water solublepolymerisation initiator to the system.

Latex or suspension polymers form the largest group of polyacrylamidesin terms of market share.

Anionic and neutral polyacrylamide polymers are readily treated bydirect addition of amidase using the process of the invention or otherprocesses, e.g. the process of our corresponding British PatentApplication No. 8630012 (ICI Case B 34139) since they are formulated tohave pH values in the range to 9. Cationic polyacrylamide polymers mayalso be treated with amidase by the process of the invention or by otherprocesses but these polymers are formulated to have acid ph values inthe range 3 to 4 and treatment by direct addition of amidase is lesseffective than in the case of anionic and neutral polymers.

According to a second aspect of the present invention we provide aprocess for the treatment of cationic polyacrylamide polymers todecompose unreacted acrylamide present therein using an amidase enzymewherein the process comprises the following steps:

(A) raising the pH of the polymer to be treated from a value in therange 3 to 3.5 to a value in the range 4 to 9; and thereafter

(B) treating the polymer with the enzyme under conditions suitable forthe enzyme to decompose the acrylamide.

The process of the second aspect of the invention can be carried outusing amidase from any source although amidase induced in strains ofMethylophilus methylotrophus is preferred. The preferred values forother parameters described in this specification also apply to theprocess of the second aspect of the invention. In step (A) the pH may beraised by any suitable means. Preferably the pH is raised to a value inthe range 4.5 to 7 and particularly to a value in the range 4.5 to 5.5or 6.

The amidase enzyme used in the decomposition and acrylic acid productionprocesses of the invention may be present in any suitable form in wholemicroorganism cells or as a crude or purified enzyme extract. The enzymecan be introduced to the processes in whole cells in the cultureproduced initially in the method of the invention or in a mediumproduced after only partial separation of water and other componentsfrom such a culture. Generally however it is preferred that the cellsshould be separated from the culture before being used in the processes.

The species Methylophilus methylotrophus (formerly named Pseudomonasmethylotropha), strains of which are cultivated in the method of theinvention, is described in our UK Patent Specification No. 1370892.Strains of this species suitable for use in the method of the inventionhave been deposited at the following 3 culture collections from whichcultures are available:

1. The National Collection of Industrial Bacteria (NCIB), TorreyResearch Station, PO Box 31, 135 Abbey Road, Aberdeen AB9 8DG, Scotland,UK;

2. The Agricultural Research Culture Collection (NRRL), 815 NorthUniversity Street, Peoria, Ill. 61604, USA; and

3. The Fermentation Research Institute (FRI), Agency of IndustrialScience and Technology, Ministry of International Trade and Industry,1 - 3 Higashi 1-Chome, Yatabe-machi, Tsukuba-gun, Ibaragi-ken, Japan.

The corresponding accession numbers assigned to the strains deposited atthese collections are as follows:

NCIB 10508-10515 and 10592-10596, all inclusive;

NRRL B 5352-B 5364 inclusive; and

FRI 1215-1227 inclusive.

A preferred strain for use in the method of the invention isMethylophilus methylotrophus strain AS-1 (NCIB 10515) which may safelybe used in treating, e.g. polyacrylamide polymers intended for use inconnection with foods. The species Methylophilus methylotrophus and theabove strains, particularly strain AS-1, have become widely known andare mentioned in numerous publications both by us and others. Inaddition to the deposits mentioned above cultures of them are also heldin a number of University and other laboratories in the UK and in othercountries. Also very suitable for use in the method of the invention isstrain NCIB 11585, available from NCIB, the production of which by thegenetic modification of strain NCIB 10515 is described in our EuropeanPatent Specification No. 35831 B and a number of other publications.

The amidase enzyme induced in Methylophilus methylotrophus has theproperties set out below.

Methylophilus methylotrophus amidase properties

    __________________________________________________________________________    Methylophilus methylotrophus amidase properties                               __________________________________________________________________________    Physiological                                                                         Inducible                                                             status                                                                        Location                                                                              Cytoplasmic                                                           Protein Soluble                                                                       Acidic  Isoelectric point                                                                         4.2                                                       Tetrameric                                                                    Native MW                                                                             (Gel permeation)                                                                          155,000                                                   Monomer MW                                                                            (SDS page)   38,000                                           Properties                                                                            pH optimum                                                                            Acetamide   5                                                                 Acrylamide  8                                                         Temperature                                                                   optimum             60° C.                                             Km      Acetamide   0.2 mM                                                            Acrylamide   10 mM                                            Activities                                                                            Acyl amidase                                                                  Acyl transferase                                                              Substrate                                                                             C.sub.1 <C.sub.2 <C.sub.3 >C.sub.4 >C.sub.5                                   Acetamide<Acrylamide>propionamide                             __________________________________________________________________________

In the method of the invention a strain of Methylophilus methylotrophusis cultivated aerobically in a medium containing sources of carbon,nitrogen, phosphorus and other appropriate nutrients with an amide beingpresent under conditions such that amidase is induced in the bacterialcells. Suitably cultivation takes place at a temperature in the range20° to 40° C., preferably 34° to 38° C., and at a pH in the range 5.0 to8.0, preferably 5.8 to 7.6. Cultivation can be by batch culture, singlestage continuous culture or multiple stage continuous culture. Asuitable dilution rate for continuous culture is in the range 0.05 hr⁻¹to 0.55 hr⁻¹. Methanol is the preferred carbon source. Any suitableamide may be present in the culture medium. Acetamide or formamide arepreferred. Other suitable amides include amides of carboxylic acidshaving the formula: ##STR3## Where R is a short chain aliphatic group,i.e. containing 1 to 5 carbon atoms, which may be a straight or abranched chain. Preferably the amide is the sole or major nitrogensource and cultivation is by continuous culture under nitrogenlimitation. A very suitable culture medium has the followingcomposition:

    ______________________________________                                        Component         Amount present/liter                                        ______________________________________                                        Phosphoric acid   1.6       ml                                                MgSO.sub.4.7H.sub.2 O                                                                           1.912     g                                                 K.sub.2 SO.sub.4  0.952     g                                                 CuSO.sub.4.5H.sub.2 O                                                                           0.840     mg                                                ZnSO.sub.4.H.sub.2 O                                                                            2.568     mg                                                MnSO.sub.4.4H.sub.2 O                                                                           4.04      mg                                                FeSO.sub.4.7H.sub.2 O                                                                           37.20     mg                                                Calcium formate   0.173     g                                                 ______________________________________                                    

Nitrogen is supplied either from acetamide alone or from acetamide andammonia. Cells are grown in nitrogen excess or nitrogen limitation at arange of cell concentrations.

Cells produced by continuous, batch or fed batch fermentation can beharvested by any suitable means preferably by ultrafiltration orcentrifugation to produce a slurry having 10-25% by weight dry solids.Suitably this slurry is broken, e.g. by several freeze-thaw cycles or bymechanical breakage in a bead mill or french pressure device. Celldebris may then be removed by centrifugation leaving a crude cell-freeextract containing amidase. Heat treatment may take place before ofafter this centrifugation but preferably before as this gives improvedsedimentation of the product during centrifugation in addition tostimulating activity. This extract can if desired be further purified byanionic ion exchange chromatography and gel filtration. The cell freeamidase preparations are suitably stored cool at 0°-10° C. or as frozensolutions or as freeze dried preparations prior to hydration and use.

When cells or cell free extracts containing amidase produced by themethod of the invention are heated to temperatures in the range 40° to80° C., as described in our co-pending British Patent Application No.8630012 (ICI Case B 34139), we have found that, most unusually, theamidase activity is irreversibly increased by 1.5 to 35 times dependentupon the enzyme preparation. This effect is greatest at temperatures inthe range 55° to 65° C., especially in the range 58° to 62° C., at pHsin the range 4 to 9, especially at pHs between 6 and 7 and at proteinconcentrations in the range 0.1 to 200 mg/ml. Under these conditionslittle significant denaturation of amidase takes place.

Heating suitably takes place for a period in the range 20 minutes to 10hours, especially 30 minutes to 180 minutes in some instances. Toproduce the increased activity the cells or cell-free enzyme can beheated immediately after they have been produced by the method of theinvention or at some later time. The method of the invention canusefully be carried out either as described above or as a similarprocess having four steps, i.e. a first fermentation step in which cellshaving induced amidase are grown in a fermenter, a second centrifugationstep wherein cells are concentrated to a 10 to 20% dry solids slurry, athird heat shock step in which cells are subjected to heating to atemperature in the ranges described above and a fourth separation stepin which a precipitate containing debris is separated from a supernatantliquid containing the enzyme.

In the process of the invention amidase can be used to reduce freeacrylamide residues occurring in all types of acrylamide polymers,particularly lattices of the three basic chemical types. When treating alatex an amidase solution is added to the latex and is dispersed throughthe latex by stirring or similar mixing techniques at a level between 1and 10,000 units/kg latex preferably 100-2000 U/kg latex. Incubation ofamidase with the latex results in conversion of free acrylamide toacrylic acid or a salt thereof. Incubation temperatures of 10° C. to100° C. (particularly 30° to 80° C.) are preferred. The latices aresuitably treated over a pH range 3 to 10, preferably 5 to 7.

The amidase produced in the method of the invention converts acrylamideinto acrylic acid. This reaction can therefore be used as a means forproducing acrylic acid or, when carried out in the presence of asuitable salt or alcohol, to produce salts or esters of acrylic acid.

The amidase produced by the method of the invention particularly whenheated as described above exhibits very high activity. Thus use of theprocess of the invention for decomposing acrylamide greatly extends therange of utility of polyacrylamide polymers.

15 The invention is illustrated by the following Examples:

EXAMPLE 1 Heat stimulation of the amidase activity of cell free extractsof Methylophilus methylotrophus AS-1.

A series of cell free extracts prepared from Methylophilusmethylotrophus AS-1 in a citrate-phosphate buffer system at pH 6.0 andat a concentration of 30 mg protein per ml were heated in a water bathfor periods of 1 and 2 hours at a series of temperatures between 30° and70° C. Samples were withdrawn at various times and were assayed foramidase activity at 30° C. The substrate used in all cases wasacrylamide at a concentration of 100 mM. The assays were performed at30° C. in a 0.1M citrate: 0.2M phosphate buffer system at pH 6.0.

The results are shown in the Table and show increased activity undermost of the conditions used with the highest increases being at 60° C.Increases were greater after 2 hours heating than after 1 hour.

                  TABLE                                                           ______________________________________                                                        Amidase                                                       Treatment       Activity (U/ml)*                                                                             Stimulation                                    ______________________________________                                        none                3.69           --                                         heating for 1 hr @                                                                        30° C.                                                                         3.69           1     x                                                50° C.                                                                         5.91           1.6   x                                                60° C.                                                                         24.65          6.7   x                                                70° C.                                                                         14.48          3.9   x                                    heating for 2 hrs @                                                                       30° C.                                                                         3.76           1.02  x                                                50° C.                                                                         8.86           2.04  x                                                60° C.                                                                         40.06          10.8  x                                                70° C.                                                                         2.28           0.62  x                                    ______________________________________                                         *1 unit is defined as 1μ mole of NH.sub.3 released per minute.        

EXAMPLE 2

Cell-free extracts as described in Example 1 were incubated at 55° C.,60° C. and 65° C. respectively. Samples were taken at various times andassayed for amidase activity as described in Example 1. The results areset out in FIG. 1 which is a graph of amidase activity (units/ml)against activation time (mins) and show optimal stimulation of amidaseactivity at 60° C. over a period of two hours. Incubation at lowertemperatures results in slower activation. Incubation at highertemperatures, e.g. at 65° C., shows an initial stimulation of rate ofactivation compared to 60° C. but a lower final amidase activity.

EXAMPLE 3 Removal of acrylamide from an anionic latex.

An anionic latex, Nalfloc type A 626 produced by the Nalfloc Co. ofCheshire, UK, pH adjusted to 5.9 at 60° C. was treated by addition of anamidase solution produced by the method of the invention of 300 unitsper ml activity to a final level of 1000 units amidase per kg latex.Samples were withdrawn at varying times and were analysed for freeacrylamide by high pressure liquid chromatography (HPLC).

The results are set out in FIG. 2 which is a graph of free acrylamide(mM) against treatment time (mins) and show a reduction in freeacrylamide level from 1.78 mM (126 ppm) to 0.02 mM (1.4 ppm) within 30minutes.

EXAMPLE 4 Removal of acrylamide from a cationic latex.

A cationic latex, Nalfloc type 4625-SC, pH adjusted to 6.0 was treatedas described in Example 3 with samples being analysed by HPLC.

The results are set out in FIG. 3 which is a graph of free acrylamide(mM) against treatment time (mins) and show a reduction in freeacrylamide level from 2.2 mM (156 ppm) to 0.02 mM (1.4 ppm) within 45minutes.

EXAMPLE 5 Removal of acrylamide from a non-ionic latex.

A non-ionic latex, Nalfloc type 8861-SC pH adjusted to 6.0 was treatedas described in Example 3 with samples being analysed by HPLC.

The results are set out in FIG. 4 which is a graph of free acrylamide(mM) against treatment time (mins) and show a reduction in freeacrylamide level from 3.1 mM (220 ppm) to 0.15 mM (10.7 ppm) within 120minutes.

EXAMPLE 6

An anionic latex Nalfloc type A.625 pH adjusted to pH 5.9 at 20° C. wastreated with amidase at a dose of 1000 units (U)/Kg A.625 as describedin Example 3.

The results are set out in FIG. 5 which is a graph of residualacrylamide (ppm BOL*) against treatment time (mins) and show a reductionin free acrylamide level from 383 ppm (5.4 mM) to 161 ppm (2.27 mM).within 390 minutes.

EXAMPLE 7

A cationic latex Nalfloc type 4625-SC was treated with amidase at a doseof 1000 units/Kg latex at 60° C. at both its native pH (˜4) and at anadjusted pH of 6. Samples were withdrawn at various times and assayedfor acrylamide content by HPLC.

The results are set out in FIG. 6 which is a graph of residualacrylamide ppm BOL against treatment time (mins) and show rapidreduction of free acrylamide in the latex adjusted to pH 6.0 but onlylimited reduction in the latex at its natural pH.

We claim:
 1. A process for the decomposition of acrylamide in a mediumcontaining it in which the medium is contacted with an enzyme capable ofdecomposing acrylamide under conditions suitable for the enzyme todecompose the acrylamide wherein the enzyme is an amidase enzyme whichhas been induced in a bacterium belonging to the species Methylophilusmethylotrophus.
 2. A process according to claim 1 wherein the amidaseenzyme has been induced in a strain selected from the group consistingof NCIB 10508 to 10515, NCIB 10592 to 10596, all inclusive, and NCIB11585 and variants and mutants of any of these strains.
 3. A processaccording to claim 1 wherein the amidase enzyme has been heated to atemperature in the range 40° to 80° C. for a sufficient period undersuitable conditions to increase the activity of the enzyme.
 4. A processaccording to claim 3 wherein the amidase enzyme has been heated to atemperature in the range 58° to 62° C.
 5. A process according to claim 3wherein the enzyme has been heated to a temperature in the range 40° to80° whilst it is contained in broken cells and/or is associated withcell debris.
 6. A process for the production of acrylic acid or a saltor ester thereof in which a medium containing acrylamide is contactedwith an enzyme capable of decomposing acrylamide to acrylic acid underconditions suitable for the enzyme to decompose the acrylamide toacrylic acid or a salt or ester thereof wherein the enzyme is an amidaseenzyme which has been induced in a bacterium belonging to the speciesMethylophilus methylotrophus.